Type 2 diabetes (T2DM) results from impaired insulin secretion, insulin resistance, and increased hepatic glucose production with deficits in insulin secretion likely the key determinant of whether T2DM develops. However, we do not understand the cause of reduced cell mass and/or a/ cell dysfunction in human T2DM. This is because models and hypotheses about T2DM or a cells generally arise from studies of rodent models or have not been confirmed in human samples. Furthermore, most studies on the T2DM human pancreas do not adequately incorporate the clinical phenotype of the patient or the disease stage and, consequently, combine profiles from different stages. In addition, previously studied T2DM pancreatic specimens have been collected in ways that do not completely allow newly available molecular analyses. To overcome these deficits in our knowledge, we have established a new infrastructure for procuring human T2DM pancreatic specimens for analysis by new technologies and isolating islets from the same pancreas. In addition, we have assembled an interdisciplinary team of scientists with human islet biology expertise and investigators who bring new technologies for tissue and cell profiling from the neuroscience and cancer arenas. Using this new infrastructure and technologies, we propose to: 1) Identify unique protein and RNA signatures in the pancreatic islets and isolated a and cells from clinically phenotyped T2DM donors of short (i.e. <5 years)- and long (i.e. >10 years)-duration with technologies such as RNA-sequencing, tissue-clearing, and single cell phenotyping. 2) Using tissue imaging mass spectrometry, identify differentially expressed lipids, proteins, and metabolites in T2DM islets. 3) Integrate molecular signatures with functional T2DM islet profiles and test the impact of candidate molecules from the lipid, metabolite, and/or mRNA/protein datasets on a and cell activity and viability. In keeping with the goals of the R24 mechanism, our discovery-based approaches will generate new resources and datasets, create new paradigms for a/ cell dysfunction in human T2DM, and foster fundamental discoveries that will not only improve our understanding of how cell dysfunction/loss occurs, but potentially lead to therapeutic interventions.